Phosphomonoester-containing compounds are an important group to be able to analyze with accuracy and sensitivity because many bioactive molecules fall into this class. These molecules include phosphomonoester forms of nucleosides (e.g., ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides), nucleoside di- and tri-phosphates, deoxynucleoside di- and tri-phosphates, dinucleotides, trinucleotides, oligonucleotides, lipids, oligosaccharides, amino acids, sugars, peptides, metabolites and drugs. High performance analysis is required because of the multiplicity, diversity, low concentrations, and adsorptive nature of many of these phosphomonoesters, making their analysis difficult.
Aryl groups such as phenyl, pyridyl and naphthyl are common parts of organic compounds. Often an aryl group in a compound is substituted with a group such as bromo, chloro, alkyl (such as methyl), alkoxy (such as methoxy), deutero or amide. This substitution modifies the chemical or physical properties of the compound to one degree or another. More than one substitutent may be present on an aryl group.
Mass spectrometry is an important technique for analyzing many chemical substances. At its best, it provides characteristic multi-analyte detection with high sensitivity and specificity. However, rarely are these advantages brought together in a single method. In general, the sensitivity of mass spectrometry is analyte and sample dependent, and varies with the conditions in the mass spectrometer. Even when conditions are optimized for each analyte of interest, and each analyte is detected under the best possible conditions, responses can vary widely for different analytes. Different analytes also tend to fragment to different degrees in the mass spectrometer. The most intense fragment may come from only a small part of the overall structure of a compound, providing little structural characterization. This failure of mass spectrometry to achieve its full analysis potential exists for all forms of mass spectrometry. This includes the common techniques of matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), and electrospray ionization mass spectrometry (ESI-MS), including MS/MS forms of these techniques. Unfortunately, phosphomonoester compounds are among the worst in their present capability of being analyzed with high performance by mass spectrometry.
Stable isotope reagents, in which a chemical reagent is enriched in a stable isotope such as deuterium, are widely used in mass spectrometry. In one use, a known amount of a stable isotope form of the analyte is added to a sample to provide a more accurate analysis of this sample based on the principle of isotope dilution. In another use, corresponding covalent labeling reagents as an isotope duo (one ordinary and one isotope-enriched) are used separately so that the target analyte is labeled in one sample with an ordinary reagent, and the same analyte in the second sample is labeled with the corresponding stable isotope reagent. The samples then can be combined prior to subsequent cleanup steps before analysis by mass spectrometry, revealing the relative amount of the target analyte in the two samples. In a third use, a given sample is reacted with a combined isotopic duo in order to convert an analyte into a pair of products that give a mass-distinctive, split signal in the mass spectrometer to enhance specificity. Stable isotope forms of test substances are also used in mass spectrometry to sort out fragmentation pathways and identify exchangeable atoms.
The sensitivity for detection of phosphomonoester compounds can be increased and made relatively uniform by labeling the compounds with a pre-existing signal group such as a fluorescent dye or radioisotope that inherently provides intensive detection properties by the corresponding detection technique for the signal group employed. For example, Giese and Wang (U.S. Pat. No. 5,512,486) introduced imidazole reagents containing pre-existing signal groups for labeling of phosphomonoester compounds to improve detection sensitivity by the designated detection technique for the pre-existing signal group. One of these reagents, containing a pre-existing fluorescent signal group, was used to convert a nucleotide into a corresponding fluorescent phosphorimidazolide, and the latter compound then was detected by fluorescence detection. The structure of the compound was confirmed by MALDI-TOF-MS. However, high sensitivity was not demonstrated by this MS technique, since the smallest amount of fluorescent phosphorimidazolide detected in the instrument was 30 picomoles. High specificity was not demonstrated since there was no isotopic duo. Multi-analyte detection was not encouraged, since several fragmentation peaks were formed by the fluorescent phosphorimidazolide product. There was no evidence that different nucleotides could give a similar response under a single set of MS conditions.
Creation of a signal group by combining an analyte with a non-signal chemical derivatization reagent is known in the field of detection by fluorescence. For example, fluorescamine and o-phthalaldehyde non-signal reagents can be reacted with amine-bearing compounds to form fluorescent products.
Characteristic, multi-analyte analysis with high sensitivity and specificity is important for phosphomonoesters because of the great number and diversity of bioactive compounds in this class. It is important to simultaneously detect known and unknown phosphomonoesters, since not all bioactive phosphomonoester compounds may have been discovered or identified, and their role needs to be sorted out both independently and relative to known phosphomonoesters. High sensitivity is critical especially for human samples which are often limited in amount, and further may have a low concentration of phosphomonoesters. High specificity is important in the analysis of phosphomonoesters to avoid false positive and false negative results.